Regulatory T cells (Tregs) are a critical sub-population of CD4+ T cells that are essential for maintaining self tolerance and limiting chronic inflammatory diseases, but also block beneficial responses, such as limiting anti- tumour immunity. We have identified a novel inhibitory cytokine, interleukin-35 (IL35) that is preferentially expressed by Tregs and is required for their maximal suppressive activity. We have recently shown that IL35 can mediate the generation of an induced regulatory T cell population, iTr35 that mediate potent suppression in vivo via IL35. Treg-mediated suppression also induces iTr35 generation in vitro and within tumors and in helminth-infected mice. iTr35 also appear to contribute to the Treg-mediated suppression in tumors. We will test the hypothesis that the regulatory triad of Tregs, IL35 and iTr35 are collectively responsible for establishing a 'regulatory milieu', and constitute key mediators of infectious tolerance. We will use a variety of model systems, with particular focus on tumors and infection-induced intestinal inflammation; sites we hypothesize are particularly relevant for IL35-mediated suppression. AIM 1: IL35 expression and function in Tregs. We will use Ebi3 and Il12a conditional/reporter mice, combined with different Foxp3 reporters to address two questions: (A) What is the pattern and kinetics of IL35 expression in Tregs? We will assess how exposure of Tregs to distinct inflammatory and disease environments alters IL35 expression in mice relative to unmanipulated mice. (B) What is the contribution of IL35 to Treg function? We will analyze mice with a Treg-restricted deletion of Ebi3, and thus IL35 production. AIM 2: Development and function of iTr35. (A) What is the frequency and kinetics of iTr35 development in vivo? We will use two complementary approaches: (i) Ebi3/Il12a expression and IL35 production analysis of Treg and non-Treg populations isolated from inflammatory lesions in Foxp3GFP mice. (ii) Analysis of lesions in triple Foxp3/Ebi3/Il12a reporter mice. (B) Are Treg-derived IL35 and/or IL10 required for iTr35 development in vivo? iTr35 development will be assessed in the presence of Tregs that lack IL35 and/or IL10. (C) What is the contribution of iTr35 to the Treg-induced regulatory milieu? (i) We will perform adoptive transfer experiments to restrict Ebi3 deletion to Foxp3-CD4+ T cells. (ii) The contribution of iTr35 to the regulatory milieu will be assessed in a new mutant mouse model in which functional iTr35 cannot develop. AIM 3: Interplay between Tregs and iTr35 in inflammatory lesions. (A) Is the maintenance and function of iTr35 dependent on Tregs? We will use acute ablation of Tregs and temporal deletion of Ebi3 in Tregs to assess their effect on iTr35 maintenance and contribution to the regulatory milieu. (B) Is the maintenance and function of Tregs dependent on iTr35? We will assess the number, function and genetic profile of Tregs in the absence of iTr35 and determine the contribution of iTr35-derived IL35 in Treg homeostasis and function. PUBLIC HEALTH RELEVANCE: Regulatory T cells (Tregs) are a critical sub-population of CD4+ T cells that are essential for maintaining self tolerance and limiting chronic inflammatory diseases, but also block beneficial responses, such as limiting anti- tumour immunity. A greater understanding of the multiple mechanisms used by Tregs to mediate their regulatory activity will lead to novel approaches for improving transplantation tolerance, curing autoimmunity and ameliorating inflammatory diseases. Furthermore, this will also lead to the development of improved vaccination strategies for chronic infections and new therapies for cancer.